Dynamics of forward and reverse transport by the glial glycine transporter, glyt1b

Abstract

Glycine is a coagonist at the N-methyl-D-aspartate receptor. Changes in extracellular glycine concentration may modulate N-methyl-D-aspartate receptor function and excitatory synaptic transmission. The GLYT1 glycine transporter is present in glia surrounding excitatory synapses, and plays a key role in regulating extracellular glycine concentration. We investigated the kinetic and other biophysical properties of GLYT1b, stably expressed in CHO cells, using whole-cell patch-clamp techniques. Application of glycine produced an inward current, which decayed within a few seconds to a steady-state level. When glycine was removed, a transient outward current was observed, consistent with reverse transport of accumulated glycine. The outward current was enhanced by elevating intracellular or lowering extracellular [Na(+)], and was modulated by changes in extracellular [glycine] and time of glycine application. We developed a model of GLYT1b function, which accurately describes the time course of the transporter current under a range of experimental conditions. The model predicts that glial uptake of glycine will decay toward zero during a sustained period of elevated glycine concentration. This property of GLYT1b may permit spillover from glycinergic terminals to nearby excitatory terminals during a prolonged burst of inhibitory activity, and reverse transport may extend the period of elevated glycine concentration beyond the end of the inhibitory burst.

Figures

FIGURE 1

Glycine transport currents in CHO cells transfected with GLYT1b. (A) 3H-glycine uptake by CHO-GLYT1b cells (black bar); in the presence of the competitive GLYT1 substrate sarcosine (300 μM, shaded bar); and the GLYT1 specific inhibitor NFPS (1 μM, open bar) (n = 6, ANOVA). (B) A whole-cell patch-clamp recording at 0 mV of GLYT1b mediated currents. Sarcosine (300 μM, shaded bar) elicits a current that is smaller in amplitude than the current induced by glycine (300 μM, black bar). (C) Glycine concentration-response curve for transport currents mediated by CHO-GLYT1b cells voltage clamped at 0 mV. Glycine transport currents were normalized to the maximal current of each cell and fitted to the Michaelis-Menten equation (n = 10). (D) Correlation between cell capacitance and peak current amplitude (n = 15).

FIGURE 2

Characteristic features of glycine transport currents measured in the presence and absence of NFPS. (A) Application of glycine (300 μM, black bar) induces an inward current that decays to a sustained current. When glycine is removed, the current rapidly decays and transiently overshoots the baseline. When glycine is coapplied with NFPS (1 μM, open bar), both the inward and overshoot currents are blocked. The internal and external Na+ concentrations are indicated. Numbers mark the (1), baseline current; (2), peak current; (3), sustained current; (4), overshoot current; and (5), NFPS block of the overshoot current. (B) Glycine (black bar) induces an inward current, but NFPS (open bar), applied in the absence of glycine, has no effect on the baseline current.

FIGURE 3

Amplitude of overshoot current is enhanced by depletion of the Na+ gradient. Extracellular application of glycine stimulates an inward current (300 μM, black bar). (A) A glycine transport current recorded with 5 mM [Na+]i and 150 mM [Na+]e (striped bar) and (B) a different cell with 50 mM [Na+]i and either 150 mM or 50 mM (shaded bar) [Na+]e. (C) Bar graph of the sustained (black bars) and overshoot (open bars) current amplitudes as a percentage of peak current recorded in various Na+ gradients (n ≥ 10, ANOVA).

FIGURE 4

Amplitude of the peak, sustained, and overshoot currents is dependent on the extracellular glycine concentration. (A) Currents stimulated by increasing [glycine]e with 50 mM [Na+]i and 50 mM [Na+]e. (B) Concentration-response curves for the peak (▪), sustained (•), and overshoot (◊) currents normalized to the Imax of the peak current and fitted to the Michaelis-Menten equation (n = 7). The EC50 values for the peak, sustained, and overshoot currents were not significantly different (repeat measure ANOVA).

FIGURE 5

Amplitude of the sustained and overshoot currents is dependent on the time of glycine application. (A) Currents stimulated by 300 mM [glycine]e with 50 mM [Na+]i and 50 mM [Na+]e applied for 5, 15, and 60 seconds (B) Bar graph of the sustained (black bars) and overshoot (open bars) current amplitudes as a percentage of peak current recorded for each of the time periods glycine was applied (n = 3; repeat measure ANOVA).

FIGURE 6

Elevating [glycine]i stimulates an outward current that can be blocked with NFPS. (A) When the glycine and [Na+]i gradients are set as described on the figure, glycine (300 μM, black bar) stimulates an inward current in 150 mM [Na+]e (striped bar) and 50 mM [Na+]e (gray bar). NFPS (1 μM, open bar) blocks a current in 50 mM [Na+]e, but does not block a current in 150 mM [Na+]e (compare the baseline current at 150 mM [Na+]e before NFPS application and then after switching from 50 mM [Na+]e to 150 mM [Na+]e as NFPS inhibition of GLYT1b persists after washout of free drug (see Fig. 2 B)). (B) Bar graph comparing the amplitude of the NFPS blocked currents in 150 mM and 50 mM [Na+]e. (n = 4, paired Student's t-test). (C) When [glycine]i is 16 mM, NFPS (1 μM, open bar) blocks a sustained outward current. (D) The amplitude of the NFPS blocked current was enhanced by elevating [Na+]i and lowering [Na+]e (n ≥ 4).

FIGURE 7

Intracellular glycine concentration-response curve for reverse transport by GLYT1b. The amplitude of the NFPS blocked reverse transport current was measured as in Fig. 6 C, in the presence of 50 mM [Na+]e and 5 mM [Na+]i, across a range of internal glycine concentrations (0–48 mM). Raw GLYT1b reverse transport currents were fitted with the Michaelis-Menten equation (n ≥ 5).

FIGURE 8

A model of glycine transport by GLYT1b. (A) Schematic showing glycine (G) and Na+ (+) binding sequentially in a narrow pore. Na+ binds before glycine to the extracellular conformation and is first to unbind from the intracellular conformation of the transporter. (B) Markov model of GLYT1b function; all reaction rates are given in s−1 unless indicated in the figure. Some reaction rates were constrained, but others were determined by fitting two separate responses to a pulse of glycine, one obtained in 50 mM [Na+]e, and the other in 150 mM [Na+]e. Unconstrained reaction rates are shown in bold italics with asterisks. (C) A typical fit obtained with the model (shaded line) of experimental data (dotted line) in two conditions, with 50 mM [Na+]i and either 150 mM or 50 mM [Na+]e. Fit parameters can be found in the Methods (fixed parameters) and Results sections (variable parameters). (D) Independent verification of the model was obtained by using the model to predict the glycine concentration-response curve with 50 mM [Na+]i and 50 mM [Na+]e (•), and with 5 mM [Na+]i and 150 mM [Na+]e (▪). The predicted curves are in good agreement with experimental results (presented in Figs. 1 C and 4 B).